The present invention relates to a method of measuring a concentration of a specific constituent in a liquid sample, more particularly a method of measuring a protein level (albumin concentration) and a sugar level (glucose concentration) of urine collected from humans or animals, and an apparatus for measuring them. The urine sugar level and urine protein level reflect the health conditions. So, an easy-to-use and accurate measuring method has been sought.
Hitherto, a urine test has been conducted by dipping a test paper impregnated with a reagent by a testing constituent such as sugar and protein into urine, and examining a color reaction of the test paper by a spectrophotometer. This test method requires different kinds of test papers for different testing constituents, and a new test paper is required for each test, thereby leading to a high running cost. There also has a limit to automatizing the process for labor saving.
When the urine test is done by this method at home, a layman should mount and replace test papers. It is not so pleasant a job and has been a block to the spread of the urine testing unit among the households.
Then, a urine test requiring no such expendable supplies as a test paper is proposed in an international patent application with a given international publication No. W097/18470. The idea of this application is that the urine sugar and urine protein levels are quantified by measuring the angle of rotation of the urine since glucose and albumin are optically active, while other urine constituents show little optical rotatory power.
When a light is propagated through a liquid containing an optically active substance, the direction of polarization rotates in proportion to the concentration of the optically active substance. It is expressed in the following equation. EQU A=L.times..alpha. (1)
where:
L: measuring optical path length PA1 A: angle of rotation [degree] PA1 .alpha.: specific angle of rotation of optically rotatory substance PA1 L: measuring optical path length PA1 A: angle of rotation [degrees] PA1 .alpha..sub.r1 (n=1, 2, . . . , N): specific angle of rotation of substance n (N: natural number) PA1 C.sub.r1 : concentration of substance n [kg/l] PA1 a.sub.r1 : specific angle of rotation of substance n
If, for example, a light with a wavelength of 589 nm is propagated 100 mm through a glucose aqueous solution of 100 mg/dl in concentration, the direction of polarization of the light rotates 50.times.10.sup.-3 degrees.
Utilizing such property, the sugar and protein levels in urine are calculated by using the above equation. Shown in Table 1 are the specific angles of rotations of aqueous solutions of glucose and albumin at 20.degree. C.
TABLE 1 Wavelength 589 nm 670 nm Glucose 50 degrees 40 degrees Albumin -60 degrees -43 degrees
In a case N kinds of optically active substances are contained in a solution, the angle of rotation of the solution is given as follows: EQU A=L.times.(.alpha..sub.1.times.C.sub.1 +.alpha..sub.2.times.C.sub.2 + . . . +.alpha..sub.N.times.C.sub.N) (2)
where:
As is evident from equation (2), the measured angle of rotation of the solution has an information about the concentrations of the plural optically active substances dissolved in the solution. That is, the angles of rotation measured of urine is a sum of the angle of rotation caused by glucose and that caused by albumin. Since the specific angle of rotation differs with the wavelength of propagated light, the angles of rotation are measured with different wavelengths of light in this method. And the sugar and protein levels in urine are calculated by simultaneous equations of equation (2).
In this method, with one wavelength of a light source, either the sugar or protein level in urine can be calculated if the concentration of the other is known. But if neither the sugar or protein level in urine is known, two or more light sources are required. Another shortcoming is that because there is not much difference between the change in specific angle of rotation of glucose which occurs with the change in light wavelength and that of albumin as shown in Table 1, no accurate determination of the sugar and protein levels in urine can be hoped for even if a plurality of light sources are used. Especially because the protein level in urine is smaller in one order of the magnitude than the sugar level, the accuracy in determination is low.